Direct Effects, Compensation, and Recovery in Female Fathead Minnows Exposed to a Model Aromatase Inhibitor

Background Several chemicals in the environment have the potential to inhibit aromatase, an enzyme critical to estrogen synthesis. Objectives The objective of this study was to provide a detailed characterization of molecular and biochemical responses of female fathead minnows to a model aromatase inhibitor, fadrozole (FAD). Methods Fish were exposed via water to 0, 3, or 30 μg FAD/L for 8 days and then held in clean water for 8 days, with samples collected at four time points during each 8-day period. We quantified ex vivo steroid production, plasma steroids, and plasma vitellogenin (Vtg) concentrations and analyzed relative transcript abundance of 10 key regulatory genes in ovaries and 3 in pituitary tissue by real-time polymerase chain reaction. Results Ex vivo 17β-estradiol (E2) production and plasma E2 and Vtg concentrations were significantly reduced after a single day of exposure to 3 μg or 30 μg FAD/L. However, plasma E2 concentrations recovered by the eighth day of exposure in the 3-μg/L group and within 1 day of cessation of exposure in the 30-μg/L group, indicating concentration- and time-dependent physiologic compensation and recovery. Concentration-dependent increases in transcripts coding for aromatase (A isoform), cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, and follicle-stimulating hormone receptor all coincided with increased E2 production and recovery of plasma E2 concentrations. Conclusions Results of this research highlight the need to consider compensation/adaptation and recovery when developing and interpreting short-term bioassays or biomarkers or when trying to predict the effects of chemical exposures based on mode of action.

[1]  B. Senthilkumaran,et al.  Unique Expression of Gonadotropin-I and -II Subunit Genes in Male and Female Red Seabream (Pagrus major) During Sexual Maturation1 , 2000, Biology of reproduction.

[2]  Steven K. Gibb.  Toxicity testing in the 21st century: a vision and a strategy. , 2008, Reproductive toxicology.

[3]  Daniel L Villeneuve,et al.  Reproductive toxicity of vinclozolin in the fathead minnow: Confirming an anti‐androgenic mode of action , 2008, Environmental toxicology and chemistry.

[4]  Daniel L Villeneuve,et al.  Ketoconazole in the fathead minnow (Pimephales promelas): Reproductive toxicity and biological compensation , 2007, Environmental toxicology and chemistry.

[5]  Daniel L Villeneuve,et al.  Transcription of key genes regulating gonadal steroidogenesis in control and ketoconazole- or vinclozolin-exposed fathead minnows. , 2007, Toxicological sciences : an official journal of the Society of Toxicology.

[6]  Gerald T Ankley,et al.  What is normal? A characterization of the values and variability in reproductive endpoints of the fathead minnow, Pimephales promelas. , 2007, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[7]  A. Elizur,et al.  Regulation of gonadotropin subunit genes in tilapia. , 2001, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[8]  G. Ankley,et al.  Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas). , 2001, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[9]  Kelly R. Munkittrick,et al.  PROTOCOL FOR MEASURING IN VITRO STEROID PRODUCTION BY FISH GONADAL TISSUE , 1995 .

[10]  Daniel L Villeneuve,et al.  Relationship between brain and ovary aromatase activity and isoform-specific aromatase mRNA expression in the fathead minnow (Pimephales promelas). , 2006, Aquatic toxicology.

[11]  G. Ankley,et al.  NMR analysis of male fathead minnow urinary metabolites: a potential approach for studying impacts of chemical exposures. , 2007, Aquatic toxicology.

[12]  W. Ge,et al.  Follistatin suppresses FSHbeta but increases LHbeta expression in the goldfish - evidence for an activin-mediated autocrine/paracrine system in fish pituitary. , 2004, General and comparative endocrinology.

[13]  W L Miller,et al.  Molecular biology of steroid hormone synthesis. , 1988, Endocrine reviews.

[14]  J. T. Sanderson,et al.  The steroid hormone biosynthesis pathway as a target for endocrine-disrupting chemicals. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[15]  Division on Earth.  Toxicity Testing in the 21st Century: A Vision and a Strategy , 2007 .

[16]  Y. Nagahama.  17 alpha,20 beta-dihydroxy-4-pregnen-3-one, a maturation-inducing hormone in fish oocytes: mechanisms of synthesis and action. , 1997, Steroids.

[17]  P. Thomas,et al.  Progestin membrane receptors involved in the meiotic maturation of teleost oocytes: a review with some new findings , 2002, Steroids.

[18]  H. van der Putten,et al.  Discrepancy Between Molecular Structure and Ligand Selectivity of a Testicular Follicle-Stimulating Hormone Receptor of the African Catfish (Clarias gariepinus)1 , 2001, Biology of reproduction.

[19]  H. Vischer,et al.  Fish FSH receptors bind LH: how to make the human FSH receptor to be more fishy? , 2005, General and comparative endocrinology.

[20]  Gerald T Ankley,et al.  Evaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas). , 2002, Toxicological sciences : an official journal of the Society of Toxicology.

[21]  S. Kajimura,et al.  cDNA Cloning of Two Gonadotropin β Subunits (GTH-Iβ and -IIβ) and Their Expression Profiles during Gametogenesis in the Japanese Flounder (Paralichthys olivaceus)☆ , 2001 .

[22]  Daniel L Villeneuve,et al.  Comparison of fathead minnow ovary explant and H295R cell-based steroidogenesis assays for identifying endocrine-active chemicals. , 2007, Ecotoxicology and environmental safety.

[23]  N. Montserrat,et al.  Effects of follicle stimulating hormone on estradiol-17 beta production and P-450 aromatase (CYP19) activity and mRNA expression in brown trout vitellogenic ovarian follicles in vitro. , 2004, General and comparative endocrinology.

[24]  N. Maclean,et al.  Autotransgenic and allotransgenic manipulation of growth traits in fish for aquaculture: a review , 2008 .

[25]  Y. Ito,et al.  Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. , 1994, Endocrine reviews.

[26]  H. Kagawa,et al.  Effects of Luteinizing Hormone and Follicle-Stimulating Hormone and Insulin-Like Growth Factor-I on Aromatase Activity and P450 Aromatase Gene Expression in the Ovarian Follicles of Red Seabream, Pagrus major1 , 2003, Biology of reproduction.

[27]  G. Ankley,et al.  Altered gene expression in the brain and liver of female fathead minnows Pimephales promelas Rafinesque exposed to fadrozole , 2008 .

[28]  Z. Yaron.  Endocrine control of gametogenesis and spawning induction in the carp , 1995 .

[29]  David O. Norris,et al.  Vertebrate endocrinology , 1983, Nature.

[30]  A. Elizur,et al.  Regulation of fish gonadotropins. , 2003, International review of cytology.

[31]  Daniel L Villeneuve,et al.  A graphical systems model to facilitate hypothesis-driven ecotoxicogenomics research on the teleost brain-pituitary-gonadal axis. , 2007, Environmental science & technology.

[32]  W. Ge,et al.  Evidence for the existence of a local activin follistatin negative feedback loop in the goldfish pituitary and its regulation by activin and gonadal steroids. , 2007, The Journal of endocrinology.

[33]  G. Ankley,et al.  Development of quantitative real-time PCR assays for fathead minnow (Pimephales promelas) gonadotropin beta subunit mRNAs to support endocrine disruptor research. , 2006, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[34]  P. Lokman,et al.  11-Ketotestosterone and IGF-I increase the size of previtellogenic oocytes from shortfinned eel, Anguilla australis, in vitro. , 2007, Reproduction.

[35]  D. Stocco.  StAR protein and the regulation of steroid hormone biosynthesis. , 2001, Annual review of physiology.

[36]  P. Swanson,et al.  Biochemistry and physiology of fish gonadotropins , 2003, Fish Physiology and Biochemistry.

[37]  P. Lokman,et al.  11-Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis. , 2001, Comparative biochemistry and physiology. Part A, Molecular & integrative physiology.

[38]  Y. Nagahama.  17α,20β-Dihydroxy-4-pregnen-3-one, a maturation-inducing hormone in fish oocytes: Mechanisms of synthesis and action , 1997, Steroids.

[39]  P. Swanson,et al.  Reproductive Biology and Endocrinology Identification of Differentially Expressed Ovarian Genes during Primary and Early Secondary Oocyte Growth in Coho Salmon, Oncorhynchus Kisutch , 2008 .

[40]  Melvin E Andersen,et al.  Dose-response modeling in reproductive toxicology in the systems biology era. , 2005, Reproductive toxicology.

[41]  W. Ge.  Roles of the activin regulatory system in fish reproduction. , 2000, Canadian journal of physiology and pharmacology.

[42]  D. Stocco,et al.  Regulation of the acute production of steroids in steroidogenic cells. , 1996, Endocrine reviews.

[43]  R. A. van den Berg,et al.  Centering, scaling, and transformations: improving the biological information content of metabolomics data , 2006, BMC Genomics.